SUB-MICRON PARTICLE
The invention relates to a sub-micron particle comprising a payload molecule and a lipid structure being surrounded by an outer layer comprising an amphiphilic copolymer. The invention extends to method of producing the sub-micron particle. The invention also encompasses pharmaceutical compositions and vaccines comprising the sub-micron particle and medical uses thereof.
The present invention relates to sub-micron particles, and in particular to sub-micron particles per se comprising a payload molecule, such as a nucleic acid, or small molecule drug. The invention extends to methods of producing the sub-micron particles, pharmaceutical compositions and vaccines comprising the sub-micron particles, and to medical uses thereof.
Vaccines are one of the most cost-effective methods to prevent infectious diseases. The World Health Organization reported that vaccinations could prevent two to three million deaths per year. Traditional vaccines involve attenuated virus or purified signature proteins of the virus. More recently, however, RNA vaccines are promising candidates due to their rapid development and low-cost manufacture. They are among global frontrunners in the race to clinical trials against COVID-19, including the already approved messenger RNA (mRNA) vaccines of Pfizer/BioNTech and Moderna, as well as the Imperial College's self-amplifying RNA (saRNA) vaccines in clinical trials. RNA vaccines have also been used preclinically for a variety of other vaccine indications, including infectious diseases such as influenza1, rabies virus2, HIV-1 3, Zika virus 4, and Ebola virus5, as well as for cancer vaccines6-9. mRNA-based vaccines typically encode the antigen of interest and contain 5′ and 3′ untranslated regions (UTRs), whereas saRNA-based vaccines encode not only the antigen but also the viral replication machinery that enables intracellular RNA amplification and abundant protein expression10. saRNA enable a large amount of antigen production from an extremely small dose (10- to 100-fold lower than mRNA) owing to intracellular replication of the antigen-encoding RNA11. The dose-sparing quality of saRNA vaccines may facilitate scale-up and manufacturing large numbers of vaccine doses.
Although mRNA and saRNA hold great promise as a new class of vaccines and therapeutics, their clinical translation and commercialization are still limited due to two big challenges. One challenge is the difficulty on intracellular delivery problems, since the RNA have: (1) insufficient endocytosis on account of its large molecular weight and negative charge which induces repulsion to cell membrane, (2) limited intracellular protein expression due to catalytic hydrolysis/enzymolysis caused by endosomal entrapment and (3) inadequate antigen loading and maturation of antigen-presenting cells (APCs).
The other major problem is that RNA is very fragile and may readily degrade in exposure environments, thus requiring RNA vaccines to be stored and transported in a very challenging cold chain. Any break of “cold chain” would decrease RNA vaccine's efficacy significantly. For example, Pfizer/BioNTech's mRNA vaccine, the world's first approved COVID-19 vaccine, is plagued by the major hurdle requiring storage at −70° C. At refrigerated temperatures of 2-8° C., the mRNA vaccine can only be stable for only 5 days (Pfizer.com, 20/11/2020). Similarly, Moderna's mRNA already approved for COVID-19 needs to be held in storage at −20° C. This makes it extremely challenging for RNA vaccines to reach the required speed and scale of deployment for vaccinations.
Lyophilization may increase the RNA stability by avoiding aqueous conditions. However, the formed ice crystal during lyophilization exhibits physical stress on the nucleic acid (sequence) and other components of the formulation, which may lead to a damage of the nucleic acid (e.g., breakage of strands, loss of supercoiling, etc.) and irreversible aggregation or precipitation of the RNA-loaded formulation. All of these will lead to an irreversibly decreased efficacy of RNA formulations and limit the rapid development of RNA vaccines and therapeutics.
Therefore, development of stable nucleic acid vaccines and therapeutics (be they saRNA, mRNA or DNA) at 2-8° C. and non-cold chain (in particular the latter) is of importance for supply, distribution and deployment, but highly challenging. Given all these challenges, it is of unprecedented urgency to develop a novel platform with low cost and safety profile for efficient nucleic acid delivery and enhanced stability, not only at refrigerated temperatures, but even at ambient temperatures.
In the past several decades, advances in bioengineering and nanotechnology have produced a number of delivery technologies. Liposomes as the widely studied RNA delivery system need to be positively charged to electrostatically trap RNA, and a certain membrane charge density threshold has been identified as a requirement to ensure efficient endosomal escape12-14. However, payloads in liposomes are easily leaked, and the lipid membrane can be disrupted when interacting with the negatively charged cell membranes. Besides, the surface charge of liposomes will affect their aggregation behaviour as well as the adsorption of serum proteins once injected in vivo. These will cause the fast clearance of RNA and reduced in vivo transfection efficiency. The use of amphiphilic diblock copolymers to generate polymer vesicles known as polymersomes is another strategy to create structures for encapsulation. Polymersomes are also studied for oligonucleotides delivery by researchers as they have higher mechanical strength and toughness than liposomes15,16. However, the synthesis of cationic polymers requires quite tricky procedures due to the complicated biological requirements. Besides, it is more difficult for efficient delivery of mRNA or saRNA due to the much bigger molecular weight and instability of RNA than oligonucleotides.
Although some reported formulations showed efficient mRNA or saRNA delivery efficacy17,18, the thermal stability is still challenging as they were used fresh or kept at −80° C. for use later.
The present invention arises from the inventors' work in attempting to overcome the problems associated with the prior art.
In accordance with a first aspect of the invention, there is provided a sub-micron particle comprising a payload molecule and a lipid structure, being surrounded by an outer layer comprising an amphiphilic copolymer.
Advantageously, the inventors were surprised to observe that the sub-micron particle may be used for efficient nucleic acid delivery (including saRNA, mRNA or DNA) and non-cold chain storage. This delivery system simultaneously addresses many if not all of the intended design requirements, including good biocompatibility, easy to manufacture, compact size, controlled surface charge, high RNA loading efficiency, endosomolytic capability, low cost and superior stability. Furthermore, the sub-micron particle may be produced using FDA-approved amphiphilic polymers (e.g., PEG-PCL) and cationic or ionizable lipids. Both materials are of low cost and readily available.
The amphiphilic copolymer forms a capsule, which serves as colloidal stable shell. The lipid structure can be self-assembled into aggregates, which are wrapped in the core of the capsule. The nucleic acid, such as RNA (e.g., mRNA or saRNA), can be encapsulated efficiently in this nanocontainer by electrostatic interaction with the cationic or ionizable lipid. The sub-micron particle provides dual protections for RNA: (1) the efficient condensation of RNA, and (2) the outer vesicular membrane, which is of higher mechanical stability and lower permeability than lipid bilayer. Compared to the current reported lipid nanoparticles, the colloidal stability of the sub-micron particles can be significantly enhanced as the mechanical strength of hydrophilic shell composed of polymer is much higher than that composed of lipid bilayer, allowing for better protection of the RNA during storage and applications.
Furthermore, advantageously, the sub-micron particles can be prepared by a one-pot method based on several minutes' mixing, stirring and solvent evaporation. The physicochemical and biological properties (e.g., particle size, surface charge, transfection efficiency and stability) of the sub-micron particles can be easily regulated by simply changing the mixing ratios.
The term “sub-micron” can be understood to mean that the particle of the invention has a largest maximum dimension of less than 1 μm. More preferably, the maximum dimension of the particle is less than 900 nm, less than 800 nm, less than 700 nm or less than 600 nm, and most preferably less than 500 nm, less than 400 nm, less than 300 nm or less than 200 nm. The sub-micron particle may have a largest maximum dimension of between 10 and 900 nm, between 20 and 800 nm, between 30 and 700 nm or between 40 and 600 nm, more preferably between 50 and 500 nm or between 60 and 400 nm, and most preferably between 80 and 300 nm or between 100 and 200 nm. The largest maximum dimension of the sub-micron particle may correspond to the Z-average size as determined using Zetasizer V instrument.
The payload molecule may be encapsulated by the lipid structure.
The payload molecule may be a biomolecule and/or an active pharmaceutical ingredient (API). The API may be a hydrophobic or hydrophilic API. The API may be a macromolecule or a small molecule. It may be appreciated that a small molecule could be considered to be a molecule with a molecular weight of less than 900 daltons. In some embodiments, a small molecule may have a molecular weight of less than 800 daltons, less than 700 daltons, less than 600 daltons, less than 500 daltons or less than 400 daltons. Similarly, a macromolecule may be considered to be a molecule with a molecular weight of at least 900 daltons.
In a preferred embodiment, the payload molecule is a biomolecule. For instance, the biomolecule may be or comprise an amino acid, a peptide, an affimer, a protein, a glycoprotein, a lipopolysaccharide, an antibody or a fragment thereof, or a nucleic acid.
The nucleic acid may be DNA, RNA or a DNA/RNA hybrid sequence. Preferably, the nucleic acid is DNA or RNA.
Most preferably, the nucleic acid is RNA. The RNA may be single stranded or double stranded. The RNA may be selected from the group consisting of: messenger RNA (mRNA); self-amplifying RNA (saRNA); antisense RNA (asRNA); RNA aptamers; interference RNA; micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); and small RNA.
Preferably, the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA). The skilled person would appreciate that self-amplifying RNAs may contain the basic elements of mRNA (a cap, 5′ UTR, 3′UTR, and poly(A) tail of variable length), but may be 20 considerably longer (for example 9-12 kb).
The nucleic acid sequence, preferably RNA, may be at least 10 bases in length, at least 20 bases in length, at least 50 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length at least 700 bases in length, at least 800 bases in length or at least 900 bases in length. In one preferred embodiment, the RNA is saRNA or mRNA.
The nucleic acid sequence, preferably RNA, and most preferably saRNA or mRNA, may be at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length at least 10000 bases in length, at least 11000 bases in length or at least 12000 bases in length.
In one embodiment, the nucleic acid sequence is at least 6000 bases in length. In one embodiment, the RNA is at least 6000 bases in length. In a preferred embodiment, the saRNA is at least 6000 bases in length.
In an alternative embodiment, the nucleic acid sequence is at least 900 bases in length. In one embodiment, the RNA is at least 900 bases in length. In a preferred embodiment, the mRNA is at least 900 bases in length.
The nucleic acid sequence, preferably RNA, and most preferably saRNA, may be between 5000 and 20000 bases in length, between 5000 and 15000 bases in length, between 5000 and 14000 bases in length, between 500o and 13000 bases in length, between 500o and 12000 bases in length, between 5000 and 11000 bases in length, between 5000 and 10000 bases in length, between 6000 and 20000 bases in length, between 6000 and 15000 bases in length, between 6000 and 14000 bases in length, between 6000 and 13000 bases in length, between 6000 and 12000 bases in length between, between 6000 and 11000 bases in length, between 6000 and 10000 bases in length, between 7000 and 20000 bases in length, between 7000 and 15000 bases in length, between 7000 and 14000 bases in length, between 7000 and 13000 bases in length, between 7000 and 12000 bases in length, between 7000 and 11000 bases in length, between 7000 and 10000 bases in length, between 8000 and 20000 bases in length, between 8000 and 15000 bases in length, between 8000 and 14000 bases in length, between 8000 and 13000 bases in length, between 8000 and 12000 bases in length, between 8000 and 11000 bases in length, between 8000 and 10000 bases in length, between 9000 and 20000 bases in length, between 9000 and 15000 bases in length, between 9000 and 14000 bases in length, between 9000 and 13000 bases in length, between 9000 and 12000 bases in length, between 9000 and 11000 bases in length or between 9000 and 10000 bases in length.
Alternatively, the nucleic acid sequence, preferably RNA, and most preferably mRNA, may be between 50 and 10000 bases in length, between 100 and 9000 bases in length, between 200 and 8000 bases in length, between 300 and 7000 bases in length, between 400 and 6000 bases in length, between 500 and 6000 bases in length, between 600 and 5000 bases in length, between 700 and 4000 bases in length, between 800 and 3000 bases in length or between 900 and 2000 bases in length.
In one embodiment, the nucleic acid sequence is between 6000 and 15000 bases in length. The nucleic acid sequence may be between 8000 and 12000 bases in length. The RNA may be between 6000 and 15000 bases in length. The RNA may be between 8000 and 12000 bases in length. Preferably, the saRNA is between 6000 and 15000 bases in length. Preferably the saRNA is between 8000 and 12000 bases in length.
In an alternative embodiment, the nucleic acid sequence is between 400 and 14000, between 500 and 10000, between 600 and 7500, between 700 and 5000, between 800 and 4000 or between 900 and 2000 bases in length. The RNA may between 400 and 14000, between 500 and 10000, between 600 and 7500, between 700 and 5000, between 800 and 4000 or between 900 and 2000 bases in length. Preferably, the mRNA is between 400 and 14000, between 500 and 10000, between 600 and 7500, between 700 and 5000, between 800 and 4000 or between 900 and 2000 bases in length.
The skilled person would appreciate that when the nucleic acid is double stranded, for example double stranded RNA, “bases in length” will refer to the length of base pairs.
The nucleic acid may encode at least a portion of a virus. The virus may be the SARS-CoV-2 virus or an influenza virus. The nucleic acid may encode a SARS-CoV-2 spike protein, more preferably a pre-fusion stabilized SARS-CoV-2 spike protein.
Alternatively, the nucleic acid may encode as the H1 hemagglutinin of the influenza virus. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is saRNA or mRNA.
The sub-micron particle preferably comprises a plurality of lipid structures. For instance, the sub-micron particle may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 lipid structures. The plurality of lipid structures may be surrounded by the outer layer comprising the amphiphilic copolymer.
The or each lipid structure may be a lipid nanoparticle or a liposome. Preferably, the or each lipid structure is a lipid nanoparticle.
The or each lipid structure may comprise a cationic or ionizable lipid. In some embodiments, the or each lipid structure may comprise a plurality of lipids. At least one of the plurality of lipids may comprise a cationic or ionizable lipid. The cationic or ionizable lipid may be a multivalent cationic lipid. The cationic or ionizable lipid may be a pH-sensitive lipid. The cationic or ionizable lipid may comprise a positively charged or ionizable nitrogen atom. The cationic or ionizable lipid may display a positive charge in an acidic solution. A solution may be understood to be acidic if it has a pH of less than 7 at 20° C., more preferably less than 6.5 at 20° C. A solution may be understood to be acidic if it has a pH of between 3.5 and 7 at 20° C. or between 4 and 7 at 20° C., more preferably between than 4.5 and 6.5 at 20° C. The cationic or ionizable lipid may be dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), an ethylphosphatidylcholine (ethyl PC), didodecyldimethylammonium bromide (DDAB), 3B-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), N4-Cholesteryl-Spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), DLin-MC3-DMA, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102).
In some embodiments, the lipid structure may comprise a cationic or ionizable lipid, such as DOTAP. The lipid structure may comprise at least 1 wt %, at least 10 wt %, at least 20 wt %, at least 30 wt %, at least 40 wt %, at least 50 wt %, at least 60 wt %, at least 70 wt %, at least 80 wt %, at least 90 wt % or at least 99 wt % cationic or ionizable lipid. In some embodiments, the lipid structure may consist of a cationic or ionizable lipid. Alternatively, the lipid structure may comprise between 1 and 99 wt % cationic or ionizable lipid, between 10 and 90 wt % cationic or ionizable lipid, between 20 and 85 wt % cationic or ionizable lipid, between 30 and 80 wt % cationic or ionizable lipid, between 40 and 75 wt % cationic or ionizable lipid, between 50 and 70 wt % cationic or ionizable lipid or between 55 and 65 wt % cationic or ionizable lipid.
In some embodiments, the lipid structure may comprise a sterol, such as cholesterol. The lipid structure may comprise at least 1 wt %, at least 5 wt %, at least 10 wt %, at least 15 wt %, at least 20 wt %, at least 25 wt %, at least 30 wt %, at least 35 wt % or at least 40 wt % sterol. The lipid structure may comprise less than 99 wt % sterol, less than 90 wt % sterol, less than 80 wt % sterol, less than 70 wt % sterol, less than 60 wt % sterol, less than 50 wt % sterol or less than 45 wt % sterol. The lipid structure may comprise between 1 and 99 wt % sterol, between 10 and 90 wt % sterol, between 15 and 80 wt % sterol, between 20 and 70 wt % sterol, between 25 and 60 wt % sterol, between 30 and 50 wt % cholesterol or between 35 and 45 wt % sterol.
In some embodiments, the lipid structure may comprise a combination of a cationic or ionizable lipid, such as DOTAP, and a sterol, such as cholesterol. The weight ratio of the cationic or ionizable lipid to the sterol may be between 1:99 and 99:1, between 90 and 90:10, between 20:80 and 85:15, between 30:70 and 80:20, between 40:60 and 75:22, between 50:50 and 70:30 or between 55:45 and 65:35. The weight ratio of the cationic or ionizable lipid to the sterol may be about 60:40.
The sub-micron particle may have an N/P molar ratio of at least 1:50, at least 1:20, at least 1:10, at least 1:5, at least 1:2, at least 1:1, at least 2:1, at least 3:1, at least 4:1 or at least 5:1, more preferably at least 6:1, at least 8:1 or at least 10:1, and most preferably at least 11:1, at least 13:1, at least 15:1, at least 16:1, at least 17:1 or at least 18:1. The sub-micron particle may have an N/P molar ratio of less than 1000:1, less than 500:1, less than 250:1, less than 100:1, less than 50:1, less than 40:1, less than 30:1, less than 28:1, less than 26:1, less than 24:1, less than 22:1, less than 21:1 or less than 20:1. The sub-micron particle may have an N/P molar ratio of between 1:50 and 1,000:1, between 1:10 and 500:1, between 1:5 and 250:1, between 1:2 and 100:1, between 1:1 and 50:1, between 2:1 and 40:1, between 5:1 and 30:1, between 8:1 and 28:1, between 10:1 and 26:1, between 12:1 and 24:1, between 14:1 and 22:1, between 16:1 and 20:1, or between 17:1 and 19:1. In preferred embodiment, the sub-micron particle has an N/P ratio of between 11:1 and 18:1. The N/P molar ratio may be understood to be the ratio between cationic amines in the lipid structure and anionic phosphates in the payload molecule.
The amphiphilic copolymer is preferably an amphiphilic block copolymer.
It may be appreciated that the amphiphilic copolymer may comprise at least one hydrophilic portion and at least one hydrophobic portion. In some embodiment, the amphiphilic copolymer comprises or consists of one hydrophilic portion and one hydrophobic portion. The or each hydrophilic portion may comprise or be a polyether, an amino acid based polymer or polypeptide, poly(2-methyloxazoline) (PMOXA), and/or a derivative thereof. The or each hydrophobic portion may comprise or be a polyester, an acid-labile polycarbonate, poly(ethylethylene) (PEE), poly(butadiene) (PBD), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(styrene) (PSt) and/or a derivative thereof.
Preferably, the amphiphilic copolymer is biodegradable. Accordingly, the or each hydrophobic portion may comprise or be a polyester, an acid-labile polycarbonate and/or a derivative thereof.
The acid-labile polycarbonate may be poly(trimethylene carbonate) (PTMC), poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate) (PTMBPEC) or a derivative thereof. The amino acid based polymer or polypeptide may be poly(L-glutamic acid) (PGA), poly-L-lysine (PLL) or a derivative thereof.
Any suitable polyether may be used. In some embodiments, the or each polyether may be polyethylene glycol (PEG), oligo(ethylene glycol) (oligoEG) or a derivative thereof. For instance, derivatives of PEG could include poly(ethylene glycol) methyl ether acrylate (mPEGA) and poly(ethylene glycol) methyl ether methacrylate (mPEGMA).
Alternatively, the polyether may be a polyether disclosed in the applicant's earlier patent application GB2009720.o. For instance, the hydrophilic portion may comprise or consist of:
appreciated that n is an integer.
Similarly, any suitable polyester may be used. In some embodiments, the or each polyester may be selected from the list consisting of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glyeolic acid) (PLGA), poly(ε-decalactone) (PDL) or a derivative thereof. Alternatively, the polyester may be a polyester disclosed in the applicant's earlier patent application GB2009720.0. For instance, the hydrophobic portion may comprise or consist of:
It may be appreciated that m and n are integers.
The hydrophilic portion may comprise less than 60 wt %, less than 50 wt %, less than 45 wt %, less than 40 wt %, less than 35 wt % or less than 32 wt % of the amphiphilic copolymer. The hydrophilic portion may comprise between 5 and 60 wt % of the amphiphilic copolymer, more preferably between 10 and 50 wt % or between 20 and 45 wt % of the amphiphilic copolymer, and most preferably between 25 and 40 wt %, between 28 and 35 wt % or between 30 and 32 wt % of the amphiphilic copolymer.
The hydrophobic portion may comprise at least 40 wt %, at least 50 wt %, at least 55 wt %, at least 60 wt %, at least 65 wt % or at least 68 wt % of the amphiphilic copolymer.
The hydrophobic portion may comprise between 40 and 95 wt % of the amphiphilic copolymer, more preferably between 50 and 90 wt % or between 55 and 80 wt % of the amphiphilic copolymer, and most preferably between 60 and 75 wt %, between 65 and 72 wt % or between 68 and 70 wt % of the amphiphilic copolymer.
The amphiphilic copolymer may have a molecular weight of at least 1,000 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da or at least 5,000 Da. Preferably, the amphiphilic copolymer has a molecular weight of at least 6,000 Da or at least 7,000 Da. In some embodiments, the amphiphilic copolymer has a molecular weight of at least 8,000 Da, at least 10,000 Da, at least 12,000 Da, at least 14,000 Da, at least 15,000 Da or at least 16,000 Da.
The amphiphilic copolymer may have a molecular weight of less than 100,000 Da, less than 80,000 Da, less than 70,000 Da, less than 60,000 Da or less than 50,000 Da. Preferably, the amphiphilic copolymer has a molecular weight of less than 40,000 Da or less than 30,000 Da. Most preferably, the amphiphilic copolymer may have a molecular weight of less than 25,000 Da, less than 20,000 Da, less than 18,000 Da, less than 17,000 Da or less than 16,000 Da.
The amphiphilic copolymer may have a molecular weight of between 1,000 and 100,000 Da, between 2,000 and 80,000, between 3,000 and 70,000, between 4,000 and 60,000, between 5,000 and 50,000, more preferably between 6,000 and 40,000, between 7,000 and 50,000, between 8,000 and 25,000, between 10,000 and 20,000, between 12,000 and 18,000, between 14,000 and 16,000 Da, between 15,000 and 17,500 Da or between 16,000 and 17,000 Da.
The hydrophobic portion may have a molecular weight of at least 1,000 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da or at least 5,000 Da. Preferably, the hydrophobic portion has a molecular weight of at least 6,000 Da or at least 7,000 Da. In some embodiments, the hydrophobic portion has a molecular weight of at least 8,000 Da, at least 10,000 Da or at least 11,000 Da.
The hydrophobic portion may have a molecular weight of between 1,000 and 70,000, between 2,000 and 60,000, between 3000 and 50,000, more preferably between 4,000 and 40,000, between 5,000 and 50,000, between 6,000 and 25,000, between 7,000 and 20,000, between 8,000 and 18,000, between 9,000 and 15,000 Da, between 10,000 and 13,000 Da or between 11,000 and 12,000 Da.
The molecular weight of the amphiphilic copolymer defined above may be understood to be the number-average molecular weight (M.).
The molecular weight of the amphiphilic copolymer, the molecular weight of the hydrophobic portion and/or the molecular weight of the hydrophilic portion may be determined using NMR or gel permeation chromatography (GPC). The methods of using NMR and GPC may be as described in the examples. In some embodiments, the molecular weight is determined using NMR, preferably 1H NMR.
The weight ratio of the amphiphilic copolymer to the payload molecule may be at least 5:1, at least 10:1, at least 20:1, at least 30:1 or at least 40:1, more preferably at least 50:1, or at least 55:1, and most preferably at least 60:1. The weight ratio of the amphiphilic copolymer to the payload molecule may be less than 1000:1, less than 500:1, less than 250:1, less than 200:1 or less than 150:1, more preferably less than 125:1, less than 100:1, and most preferably less than 85:1. The weight ratio of the amphiphilic copolymer to the payload molecule may be between 5:1 and 1000:1, between 10:1 and 500:1, between 20:1 and 250:1, between 30:1 and 200:1 or between 40:1 and 150:1, more preferably between 50:1 and 125:1, or between 55:1 and 100:1, and most preferably between 60:1 and 85:1.
The weight ratio of the amphiphilic copolymer to the cationic or ionizable lipid may be at least 1:10, at least 1:8, at least 1:6, at least 1:4, at least 1:2 or at least 1:1.5, more preferably at least 1:1, at least 1.5:1, at least 1.75:1, at least 2:1, at least 2.2:1 or at least 2.4:1, and most preferably at least 2.5:1. The weight ratio of the amphiphilic copolymer to the cationic or ionizable lipid may be less than 50:1, less than 20:1, less than 15:1, less than 10:1, less than 8:1 or less than 6:1, more preferably less than 5.5:1, less than 5:1, less than 4.5:1, less than 4:1, less than 3.5:1 or less than 3:1, and most preferably less than 2.7:1. The weight ratio of the amphiphilic copolymer to the cationic or ionizable lipid may be between 1:10 and 50:1, between 1:8 and 20:1, between 1:6 and 15:1, between 1:4 and 10:1, between 1:2 and 8:1 or between 1:1.5 and 6:1, more preferably between 1:1 and 5.5:1, between 1.5:1 and 5:1, between 1.75:1 and 4.5:1, between 2:1 and 4:1, between 2.2:1 and 3.5:1 or between 2.4:1 and 3:1, and most preferably between 2.5:1 and 2.7:1.
The outer layer comprising the amphiphilic copolymer preferably comprises a thickness of at least 0.5 nm, at least 1 nm or at least 1.5 nm, more preferably at least 2 nm or at least 2.5 nm, and most preferably at least 3 nm. The outer layer comprising the amphiphilic copolymer preferably comprises a thickness of less than 25 nm, less than nm or less than 15 nm, more preferably less than 10 nm or less than 7.5 nm, and most preferably less than 5 nm. The outer layer comprising the amphiphilic copolymer preferably comprises a thickness of between 0.5 to 25 nm, between 1 to 20 nm or between 1.5 to 15 nm, more preferably between 2 to 10 nm or between 2.5 to 7.5 nm, and most preferably between 3 to 5 nm.
The sub-micron particle may further comprise at least one stabilizing molecule. The at least one stabilizing molecule may be surrounded by the outer layer comprising the amphiphilic copolymer. Alternatively, or additionally, the at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer.
The weight ratio of the stabilizing molecule to the payload molecule may be at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1 or at least 95:1. In some embodiments, the weight ratio of the stabilizing molecule to the payload molecule may be at least 100:1, at least 250:1, at least 500:1, at least 1,000:1, at least 2,000:1, at least 3,000:1, at least 4,000:1 or at least 5,000:1. The weight ratio of the stabilizing molecule to the payload molecule may be less than 100,000:1, less than 50,000:1, less than 10,000:1, less than 9,000:1, less than 8,000 to 1, less than 7,000:1, or less than 6,500:1. In some embodiments, the weight ratio of the stabilizing molecule to the payload molecule may be less than 5,000:1, less than 1,000:1, less than 500:1, less than 400:1, less than 350:1, less than 300:1, less than 250:1, less than 200:1, less than 175:1, less than 150:1, less than 125:1 or less than 110:1. The weight ratio of the stabilizing molecule to the payload molecule may be between 1:1 and 100,000:1, between 2:1 and 50,000:1, between 4:1 and 10,000:1, between 6:1 and 9,000:1, between 8:1 and 8,000:1, between 10:1 and 7,000:1 or between 20:1 and 6,500:1. In some embodiments, the weight ratio of the stabilizing molecule to the payload molecule may be between 6:1 and 5,000:1, between 8:1 and 1,000:1, between 10:1 and 500:1, between 20:1 and 400:1, between 30:1 and 350:1, between 40:1 and 300:1, between 50:1 and 250:1, between 60:1 and 200:1, between 70:1 and 175:1, between 80:1 and 150:1, between 90:1 and 125:1 or between 95:1 and 110:1. In alternative embodiments, the weight ratio of the stabilizing molecule to the payload molecule may be between 90:1 and 100,000:1, between 100:1 and 50,000:1, between 500:1 and 25,000:1, between 1,000:1 and 10,000:1, between 2,000:1 and 9,000:1, between 3,000:1 and 8,000:1, between 4,000:1 and 7,000:1 or between 5,000:1 and 6,500:1. These weight ratios may relate to the total weight of the stabilizing molecule to the payload molecule, i.e. they may include the weight of any stabilizing molecules which are surrounded by the outer layer and any stabilizing molecules which are disposed outside the outer layer.
In embodiments where the at least one stabilizing molecule is surrounded by the outer layer comprising the amphiphilic copolymer may be encapsulated in the lipid structure. Alternatively, or additionally, the at least one stabilizing molecule surrounded by the outer layer comprising the amphiphilic copolymer may be disposed outside the lipid structure. In embodiments where the sub-micron particle comprises a plurality of lipid structures, at least one stabilizing molecule may be disposed between the plurality of lipid structures.
The weight ratio of the stabilizing molecule surrounded by the outer layer to the payload molecule may be at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1 or at least 95:1. The weight ratio of the stabilizing molecule surrounded by the outer layer to the payload molecule may be less than 100,000:1, less than 50,000:1, less than 10,000:1, less than 5,000:1, less than 1,000:1, less than 500:1, less than 400:1, less than 350:1, less than 300:1, less than 250:1, less than 200:1, less than 175:1, less than 150:1, less than 125:1 or less than 110:1. The weight ratio of the stabilizing molecule surrounded by the outer layer to the payload molecule may be between 1:1 and 100,000:1, between 2:1 and 50,000:1, between 4:1 and 10,000:1, between 6:1 and 5,000:1, between 8:1 and 1,000:1, between 10:1 and 500:1, between 20:1 and 400:1, between 30:1 and 350:1, between 40:1 and 300:1, between 50:1 and 250:1, between 60:1 and 200:1, between 70:1 and 175:1, between 80:1 and 150:1, between go:1 and 125:1 or between 9s:1 and 110:1.
Alternatively or additionally, at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer. The at least one stabilizing molecule disposed outside the outer layer comprising the amphiphilic copolymer may be disposed at a concentration of at least 1 mg/ml, at least 5 mg/ml, at least 10 mg/ml or at least 50 mg/ml, more preferably at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml or at least 220 mg/ml, and most preferably at least 240 mg/ml. The at least one stabilizing molecule disposed outside the outer layer comprising the amphiphilic copolymer may be disposed at a concentration of less than 100,000 mg/ml, less than 50,000 mg/ml, less than 10,000 mg/ml, less than 5,000 mg/ml or less than 1,000 mg/ml, more preferably less than 750 mg/ml, less than 500 mg/ml, less than 300 mg/ml or less than 280 mg/ml, and most preferably less than 260 mg/ml. The at least one stabilizing molecule disposed outside the outer layer comprising the amphiphilic copolymer may be disposed at a concentration of between 1 and 100,000 mg/ml, between 5 and 50,000 mg/ml, between 10 and 10,000 mg/ml, between 25 and 5,000 mg/ml or between 50 and 1,000 mg/ml, more preferably between 100 and 750 mg/ml, between 150 and 500 nm/ml, between 200 and 300 nm/ml, or between 220 and 280 nm/ml, most preferably between 240 and 260 mg/ml.
In a preferred embodiment, the sub-micron particle comprises at least one stabilizing molecule surrounded by the outer layer comprising the amphiphilic copolymer and at least one stabilizing molecule is disposed outside the outer layer comprising the amphiphilic copolymer.
The or each stabilizing molecules may be a carbohydrate and/or a polyol.
The carbohydrate may be referred to as a sugar. The carbohydrate may be a monosaccharide, which may be selected from a group consisting of: glucose; galactose; fructose; mannose; and xylose or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. Alternatively, the carbohydrate may be a disaccharide, which may be selected from a group consisting of: trehalose; sucrose; lactose; maltose; isomaltose; lactitol; lactulose; mannobiose; and isomalt or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In a further alternative, the carbohydrate may be a trisaccharide, which may be selected from a group consisting of: nigerotriose; maltotriose; melezitose; maltotriulose; raffinose; and kestose or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. In a further alternative, the carbohydrate may be a polysaccharide, which may be selected from the group consisting of: dextran; amylose; amylopectin; glycogen; galactogen; inulin; callose; cellulose; chitosan; and chitin or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.
The carbohydrate may be a polyol, which may be selected from a group consisting of: sorbitol; mannitol; glycerol; alpha-D-glucopyranosyl-1-6-sorbitol; alpha-D-glucopyranosyl-1-6-mannitol; a malto-oligosaccharide; a hydrogenated maltooligosaccharide, starch and cellulose or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.
Alternatively or additionally, the polyol may be an oligomer comprising a plurality of hydroxyl groups; a polymer comprising a plurality of hydroxyl groups or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.
The at least one stabilizing molecule may comprise at least two different stabilizing molecules. Each stabilizing molecule may be a carbohydrate. In one embodiment, a first stabilizing molecule may be a disaccharide (e.g., trehalose) and a second stabilizing molecule may be a polysaccharide (e.g., dextran).
In some embodiments, the carbohydrate is a disaccharide, and most preferably trehalose, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof. The trehalose may be synthetic trehalose or natural trehalose.
The sub-micron particle may comprise at least one targeting ligand or moiety. The at least one targeting ligand or moiety may be disposed on an outer surface of the sub-micron particle. Accordingly, the at least one targeting ligand or moiety may be disposed on an outer surface of the outer layer comprising the amphiphilic copolymer.
The at least one targeting ligand or moiety may be or comprise at least one of a peptide, a protein, an aptamer, a carbohydrate, an oligosaccharide, a folic acid or folate, and antibody or an antigen binding fragment thereof, a vitamin or a derivative thereof. The peptide may be a G protein-coupled receptor (GCR), Arg-Gly-Asp (RGD), or a derivative thereof. The proteins may be a lectin, a transferrin, or a derivative thereof.
The aptamers may be an RNA aptamer against HIV glycoprotein, or a derivative thereof. The carbohydrates may be as defined above. In particular, the carbohydrate may be mannose, glucose, galactose, or a derivative thereof. The antibody may be monoclonal or polyclonal. The antibody may be an anti-Her2 antibody, an anti-EGFR antibody, or a derivative thereof. The vitamin may be vitamin D.
The sub-micron particle may be stored in solution. The solution may be an aqueous solution. The sub-micron particle may be present at a concentration such that the concentration of the payload molecule is at least 0.001 μg/ml, at least 0.01 μg/ml, at least 0.05 μg/ml, at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 15 μg/ml or at least 20 μg/ml. The sub-micron particle may be present at a concentration such that the concentration of the payload molecule is less than 500 mg/ml, less than 100 mg/ml, less than 10 mg/ml, less than 5 mg/ml, less than 1 mg/ml, less than 500 μg/ml, less than 200 μg/ml, less than 100 μg/ml, less than 50 μg/ml or less than 30 μg/ml. The sub-micron particle may be present at a concentration such that the concentration of the payload molecule is between 0.001 μg/ml and 500 mg/ml, between 0.01 μg/ml and 100 mg/ml, between 0.05 μg/ml and 50 mg/ml, between 0.1 μg/ml and 10 mg/ml, between 0.5 μg/ml and 5 mg/ml, between 1 μg/ml and 1 mg/ml, between 5 and 500 μg/ml, between 10 and 200 μg/ml, between and 100 μg/ml, between 20 and 50 μg/ml or between 20 and 30 μg/ml.
Alternatively, the sub-micron particle may be freeze dried.
Preferably, the sub-micron particle of the first aspect is thermally stabilized.
It will be appreciated that the expression “thermal stabilization” or “thermally stabilized” can mean that the sub-micron particle substantially retains its biological activity (e.g., it elicits an immune response and/or protein expression in a subject administered therewith) when stored at certain temperatures for a period of time. Although the inventors do not wish to be bound by any hypothesis, they believe that the thermal stabilization effects may be realised by stabilizing the lipid structure in the formulation, for example by preventing or reducing its aggregation; by stabilizing the payload molecule (preferably RNA) per se; and/or by stabilizing the sub-micron particle to have improved colloidal stability. Whether or not the functional activity of the payload molecule (preferably RNA) is retained, or the extent thereof, can be determined for example by detecting the presence of immunospecific antibodies (e.g., IgG) raised against the antigen of interest encoded by the RNA construct and/or detecting the expression of a protein of interest.
Preferably, the sub-micron particle is thermally stabilised following storage at a temperature of −100° C. and above, −80° C. and above, −60° C. and above, −40° C. and above or −20° C. and above, more preferably −15° C. and above, and most preferably −10° C. and above. Preferably, the sub-micron particle is thermally stabilised following storage at a temperature of −5° C. and above, more preferably ° C. and above, and most preferably 1C and above. Most preferably, the sub-micron particle is thermally stabilised following storage at a temperature of 2° C. and above, more preferably 3° C. and above, and most preferably 4° C. and above. Even more preferably, the sub-micron particle is thermally stabilised following storage at a temperature of 5° C. and above, more preferably 6° C. and above, and most preferably 7° C. and above.
The sub-micron particle may be thermally stabilised following storage at a temperature of less than 100° C., less than 80° C., less than 60° C., less than 50° C., less than 40° C., less than 35° C., or less than 30° C. The sub-micron particle may be thermally stabilised following storage at a temperature of less than 25° C., less than 20° C., or less than 15° C. The sub-micron particle may be thermally stabilised following storage at a temperature of less than 100° C., less than 8° C., or less than 7° C.
The sub-micron particle may be thermally stabilised following storage at a temperature of between −100° C. and 100° C., between −80° C. and 90° C., between −60° C. and 80° C., between −40° C. and 70° C., between −20° C. and 60° C., between −20° C. and 50° C., between −20° C. and 40° C., between −20° C. and 35° C., between −2° C. and 30° C., between −15° C. and 25° C., or between −10° C. and 20° C. The sub-micron particle may be thermally stabilised following storage at a temperature of between −5° C. and 15° C., between 0° C. and 10° C., between 1° C. and 9° C., or between 2° C. and 8° C.
In accordance with a second aspect, there is provided a method of producing a sub-micron particle, the method comprising contacting a payload molecule, a cationic or ionizable lipid, and an amphiphilic copolymer to produce the sub-micron particle.
Advantageously, the method provides a one-pot method for providing the sub-micron particle of the first aspect.
Preferably, the payload molecule, the cationic or ionizable lipid, and the amphiphilic copolymer are contacted simultaneously. The payload molecule, cationic or ionizable lipid and amphiphilic copolymer may be understood to be contacted simultaneously if they are all present in the same reaction mixture.
The payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer may be as defined in relation to the first aspect. Furthermore, the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer may be provided at the ratios defined in relation to the first aspect.
The amphiphilic copolymer may be synthesized using any method known in the art. For instance, the amphiphilic copolymer may be synthesized using the method defined in the applicant's earlier patent application, GB2009720.0. However, it will be appreciated that alternative methods may be used.
The method may comprise providing a first solution comprising the cationic or ionizable lipid and the amphiphilic copolymer. The first solution may comprise an organic solvent. The organic solvent may be an ether, an alcohol or a nitrile. The ether may be a cyclic ether. The organic solvent may be tetrahydrofuran (THF), ethanol, methanol and acetronitrile.
The method may comprise providing a second solution comprising the payload molecule. The second solution may comprise water, preferably ribonuclease (RNase)-free water.
Contacting the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer may comprise combining the first and second solutions to produce a reaction mixture, and thereby contacting the payload molecule, the cationic or ionizable lipid, and the amphiphilic copolymer.
The method may comprise stirring the reaction mixture.
The method may comprise contacting the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer for at least 15 seconds, at least 30 seconds, at least seconds or at least 1 minute. The method may comprise contacting the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer for between 15 seconds and 30 minutes, between 30 seconds and 10 minutes, between 45 seconds and 5 minutes or between 1 and 2 minutes.
The method may comprise contacting the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer at a temperature between ° C. and 75° C., between 5° C. and 50° C., between 10° C. and 30° C. between 15° C. and 25° C. or between 19° C. and 21° C.
The method may subsequently comprise removing the organic solvent. The organic solvent may be removed by rotary evaporation.
Contacting the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer, may comprise contacting the payload molecule, the cationic or ionizable lipid, the amphiphilic copolymer and a stabilizing molecule. The payload molecule, the cationic or ionizable lipid, the amphiphilic copolymer and the stabilizing molecule may be contacted simultaneously.
Advantageously, in the resultant sub-micron particle the stabilizing molecule will be surrounded the outer layer comprising the amphiphilic copolymer.
The stabilizing molecule may be as defined in relation to the first aspect.
In embodiments where the method comprises providing a second solution, the second solution may further comprise the stabilizing molecule.
The weight ratio of the stabilizing molecule to the payload molecule in the second solution and/or in the reaction mixture may be at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1 or at least 95:1. The weight ratio of the stabilizing molecule to the payload molecule in the second solution and/or in the reaction mixture may be less than 100,000:1, less than 50,000:1, less than 10,000:1, less than 5,000:1, less than 1,000:1, less than 500:1, less than 400:1, less than 350:1, less than 300:1, less than 250:1, less than 200:1, less than 175:1, less than 150:1, less than 125:1 or less than 110:1. The weight ratio of the stabilizing molecule to the payload molecule in the second solution and/or in the reaction mixture may be between 1:1 and 100,000:1, between 2:1 and 50,000:1, between 4:1 and 10,000:1, between 6:1 and 5,000:1, between 8:1 and 1,000:1, between 10:1 and 500:1, between 20:1 and 400:1, between 30:1 and 350:1, between 40:1 and 300:1, between 50:1 and 250:1, between 60:1 and 200:1, between 70:1 and 175:1, between 80:1 and 150:1, between 90:1 and 125:1 or between 95:1 and 110:1.
Alternatively, or additionally, the method may comprise contacting the resultant sub-micron particle and a stabilizing molecule. The stabilizing molecule may be as defined in relation to the first aspect. Accordingly, at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer.
The method may comprise contacting the resultant sub-micron particle and the stabilizing molecule subsequent to removing the organic solvent. The method may comprise contacting the resultant sub-micron particle and the stabilizing molecule at a concentration to obtain the concentration of the stabilizing molecule defined in relation to the first aspect.
The method may comprise storing the sub-micron particle in solution. The solution may be as defined in relation to the first aspect.
Alternatively, the method may comprise drying the sub-micron particle. Preferably, drying the sub-micron particle comprise freeze drying the sub-micron particle.
In accordance with a third aspect, there is provided a sub-micron particle obtained or obtainable by the method of the second aspect.
In a fourth aspect, there is provided a pharmaceutical composition comprising the sub-micron particle of the first or third aspect and a pharmaceutically acceptable vehicle.
In a fifth aspect, there is provided a method of preparing the pharmaceutical composition according to the fourth aspect, the method comprising contacting the sub-micron particle of the first or third aspect with a pharmaceutically acceptable vehicle.
In a sixth aspect, there is provided the sub-micron particle of the first or third aspect, or the pharmaceutical composition of the fourth aspect, for use as a medicament.
In a seventh aspect, there is provided a method of treatment, the method comprising administering, or having administered, to a subject in need thereof, a therapeutic amount of the sub-micron particle of the first or third aspect, or the pharmaceutical composition of the fourth aspect.
In an eighth aspect, there is provided a vaccine composition comprising the sub-micron particle of the first or third aspect, or the pharmaceutical composition of the fourth aspect.
The vaccine may comprise a suitable adjuvant.
The vaccine may be a vaccine for COVID-19. The vaccine may be a vaccine for influenza virus.
In a ninth aspect, there is provided the sub-micron particle of the first or third aspect, the pharmaceutical composition of the fourth aspect or the vaccine of the eighth aspect, for use in stimulating an immune response in a subject.
The immune response may be stimulated against a protozoa, bacterium, virus, fungus or cancer. The virus may be COVID-19. The virus may be influenza virus.
In a tenth aspect of the invention, there is provided a method of vaccinating a subject, the method comprising administering, or having administered, to a subject in need thereof, a therapeutic amount of the sub-micron particle of the first or third aspect, the pharmaceutical composition of the fourth aspect or the vaccine of the eighth aspect.
The sub-micron particle, the pharmaceutical composition or the vaccine of the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.
The sub-micron particle, the pharmaceutical composition or the vaccine of the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site.
In a preferred embodiment, however, medicaments according to the invention may be administered to a subject by injection into the blood stream, muscle, skin or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intramuscular (bolus or infusion), intrathecal (bolus or infusion), epidural (bolus or infusion) or intraperitoneal (bolus or infusion).
It will be appreciated that the amount of sub-micron particle, the pharmaceutical composition or the vaccine that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the sub-micron particle, the pharmaceutical composition or the vaccine and whether it is being used as a monotherapy or in a combined therapy.
The frequency of administration will also be influenced by the half-life of the active agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the sub-micron particle, the pharmaceutical composition or the vaccine in use, the strength of the pharmaceutical composition, the mode of administration, and the type of treatment. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
The required dose may depend upon a number of factors including, but not limited to, the active agent being administered, the disease being treated and/or vaccinated against, the subject being treated, etc.
Generally, a dose of between 0.001 μg/kg of body weight and 10 mg/kg of body weight, or between 0.01 μg/kg of body weight and 1 mg/kg of body weight, of the sub-micron particle, the pharmaceutical composition or the vaccine of the invention may be used, depending upon the active agent used. A dose may be understood to relate to the quantity of the payload molecule which is delivered.
Doses may be given as a single administration (e.g., a single injection). Alternatively, the sub-micron particle, the pharmaceutical composition or the vaccine may require more than one administration. As an example, the sub-micron particle, the pharmaceutical composition or the vaccine may be administered as two or more doses of between 0.07 μg and 700 mg (i.e., assuming a body weight of 70 kg). Alternatively, a slow-release device may be used to provide optimal doses of the sub-micron particle, the pharmaceutical composition or the vaccine according to the invention to a patient without the need to administer repeated doses. Routes of administration may incorporate intravenous, intradermal subcutaneous, intramuscular, intrathecal, epidural or intraperitoneal routes of injection.
Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g., in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the sub-micron particle, the pharmaceutical composition or vaccine according to the invention and precise therapeutic regimes (such as doses of the agents and the frequency of administration).
A “subject” may be a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g., a horse), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human being.
A “therapeutically effective amount” of the sub-micron particle, the pharmaceutical composition or the vaccine is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to produce a therapeutic effect.
For example, a therapeutically effective amount of the sub-micron particle, the pharmaceutical composition and the vaccine of the invention may comprise from about 0.001 mg to about 800 mg of the payload molecule, and preferably from about 0.01 mg to about 500 mg of the payload molecule.
A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder, a capsule or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g., sub-micron particle of the invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
Alternatively, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The sub-micron particle according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and subcutaneous injection. The sub-micron particle of the invention may be prepared as any appropriate sterile injectable medium.
The sub-micron particle may be administered by inhalation. For instance, the sub-micron particle may be provided in the form of an aerosol.
The sub-micron particle and/or the pharmaceutical composition of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
The sub-micron particle of the invention and/or the pharmaceutical composition according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
All features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:-
PEG-PCL copolymers with different molecular weights were synthesized by ring-opening polymerization of ε-caprolactone (ε-CL), which was initiated by mPEG-OH using stannous octoate (Sn(Oct)2) as catalyst. Briefly, 200 mg mPEG5k-OH, 470 mg 8-CL (or 200 mg mPEG2k-OH, 530 mg ε-CL) and 50 mg Sn(Oct)2 were dissolved in 5 mL anhydrous toluene, and the reaction system was heated to 110° C. under dry nitrogen atmosphere for 48 h22. After the reaction, the mixture was degassed and cooled to room temperature. Then the resulting product was precipitated using excess cold diethyl ether. The polymer was filtered and vacuum dried to constant weight at room temperature.
Characterization of PEG-PCL copolymers 1H-NMR spectra (in CDCl3) were recorded on a Jeol 400 MHz NMR spectrometer to characterize the chemical compositions and the degree of polymerization of the PCL block. Gel permeation chromatography (GPC) was employed using the Agilent 1260 Infinity II to determine the polymer molecular weights and molecular weight distributions.
Results and Discussion
PE-LNPs were prepared with a simple one-pot method. Typically, 650 μg PEG5k-PCL10k and 250 μg DOTAP were co-dissolved in 0.5 mL THF, while 10 μg saRNA was dissolved in 1 mL RNase-free water. The organic and aqueous solutions were quickly mixed and the mixture was kept stirring for 1-2 min at room temperature. The saRNA-loaded PE-LNP 11-65 (N/P=11, polymer/saRNA (w/w)=65) were then obtained after removing THF by rotary evaporation. Various PE-LNPs with different N/P molar ratios or polymer/saRNA weight ratios were prepared similarly. All tips and glassware should be treated prior to use to ensure RNase-free conditions.
Characterization of PE-LNPsSize distribution (Z-average) and zeta potential (based on the Smulochowski model) of different PE-LNPs were determined at 25° C. using the Zetasizer μV instrument (Malvern, UK) and ZETA PALS, respectively. The thermal stability of PE-LNPs was evaluated by measuring their size and polydispersity index (PDI) upon temperature ramp from 25° C. to 85° C. through high-throughput dynamic light scattering (HT-DLS) using a DynaPro Plate Reader III (Wyatt, UK).
SEM was used to visualize the morphology of PE-LNPs. One drop of PE-LNP suspension was placed on a graphite surface. The sample was coated with gold using an Ion Sputter after drying. Afterwards, samples were viewed using a JSM-6400 scanning electron microscope (JEOL Ltd, Tokyo, Japan) at an accelerating voltage of 20 kV.
PE-LNP samples for Cryo-TEM (Tecnai F20 G2 at 200 kV) were prepared on a lacey carbon-coated copper grid (Structure Probe Incorporation, PA) using a semi-automated Vitrobot system (Vitrobot Mark II, FEI). Briefly, 4 μL of 1 mg mL−1 PE-LNP solution was casted on top of a carbon grid. The grid was then transferred to a Vitrobot chamber that was at 100% humidity and at 20° C. Rapid immersion of the grid into liquid ethane after 1 s blotting effectively vitrified the sample. The sample was kept under −170° C. using a Gatan 626 cryo holder until successfully transferred into the Cryo-TEM instrument in order to prevent crystalline ice formation. The images were obtained at a defocus of ˜4,000 nm.
The saRNA encapsulation efficiency of PE-LNPs was determined by RiboGreen Assay (Quant-iT™ RiboGreen™ RNA Assay Kit, Thermo Fisher). TE buffer and aqueous RiboGreen working solution were prepared with and without 0.5% Triton X-100 following the manufacturer's instructions. The calibration curve of fluorescence intensity versus saRNA concentration was established. The free (unloaded) saRNA concentration (Cunloaded) was determined as follows: samples were diluted with Triton X-100 free TE buffer to an appropriate concentration, mixed with Triton X-100 free RiboGreen working solution and incubated for 15 minutes in the dark. The samples were then pipetted to a black 96-well plate with each well containing 200 μL final mixture. Fluorescence intensity measurements were then taken by a spectrofluorometer (GloMax@ Discover Microplate Reader, Promega, USA) at the excitation and emission wavelengths of 480 and 520 nm, respectively, with three replicates. The total saRNA concentration (Cota) was measured following a similar procedure with TE buffer and RiboGreen working solution containing 0.5% Triton X-100 to lyse the nanoparticles. The encapsulation efficiency of the system could be calculated according to the following equation:
Various saRNA-loaded PE-LNPs with different N/P molar ratios and polymer/saRNA (w/w) weight ratios were obtained. The PE-LNP with N/P=5 and polymer/saRNA (w/w)=65 is named as PE-LNP 5-65. The other PE-LNPs are assigned according to the same rule. The size distribution, PDI and zeta potential of PE-LNPs with different compositions have been summarized in
The N/P molar ratio is a critical factor controlling the saRNA loading capacity and endosomal escape efficiency. With increasing the N/P ratio from 5 to 18, the zeta potential of the PE-LNPs composed of PEG5k-PCL10k (polymer/saRNA weight ratio=65) increased significantly from +29.0 f 0.9 mV to +44.5 f 0.8 mV due to the increased amount of the cationic lipid. However, when the polymer/saRNA weight ratio in the PE-LNPs increased from 65 to 80, zeta potential of the PE-LNPs composed of PEG5k-PCL10k (N/P=18) decreased from +44.5±0.8 mV to +40.2 f 0.9 mV, which could be due to the enhanced shielding effect of the polymer capsule layer. The PE-LNPs composed of PEG2k-PCL5k showed the same trends.
PEG5k-PCL10k and DOTAP were used to prepare the RNA-loaded PE-LNP m-n for further investigation in the exemplified work unless specified, with the numbers m and n denoting to the N/P molar ratio and polymer/RNA weight ratio, respectively.
The counterpart of PE-LNP m-n without the payload is named as PE-LNP m‘-n’. The structure of PE-LNPs without RNA loading was investigated by Cryo-TEM. The Cryo-TEM micrographs in
Accordingly, DOTAP self-assembled into “sponge-like” aggregates, which were encapsulated into the aqueous core of the polymersome and divided the internal space into sub-compartments though hydrophobic interactions of the hydrocarbon tails. Compared with liposomes and polymersomes, the internal structure of PE-LNPs provides a significantly higher surface area which is favorable for nucleic acid loading. Further, the PEG-PCL shell can not only prevent the degradation of RNA but also increase the stability of the PE-LNP system due to its higher mechanical strength than the lipid bilayer.
The inventors evaluated the thermal stability of PE-LNP 11′-65′ 11′-80′, 18′-65′ and 18′-80′. As shown in
FRET experiments were carried out to further investigate the PE-LNP structure using a fluorometer (FluoroMax-4, Horiba Scientific). For the donor/acceptor pair of N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE)/N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Rhod-PE), the excitation wavelength was set at 460 nm and emission spectra were collected from 480 to 630 nm.
PE-LNPs were incorporated with the donor NBD-PE (4 mM) and the acceptor Rhod-PE (4 mM). Briefly, for various PE-LNPs such as PE-LNP 5′-65′, PE-LNP 11′-65′ and PE-LNP 18′-65′ without saRNA loading, NBD-PE and Rhod-PE were co-dissolved in 0.5 mL THF (containing pre-dissolved PEG-PCL and DOTAP) at the desired ratio, followed by quick mixing with 1 mL H2O. The mixture was stirred for 1-2 min at room temperature and THF was then removed by rotary evaporation (see the PE-LNPs preparation section in Example 2 above). Control groups without polymer (i.e., DOTAP liposomes) were prepared for comparison. The molar concentrations of DOTAP in the control liposomes were either the same as the molar concentration of DOTAP (or the total molar concentration of both PEG-PCL and DOTAP) in the corresponding PE-LNPs. For example, the molar concentrations of DOTAP and PEG5k-PCL10k in PE-LNP 5′-65′ were 143 mM and 43 mM, respectively. Therefore, the corresponding control liposomes were denoted as DOTAP-143 and DOTAP-186, respectively. In addition, the respective donor-labelled (NBD-PE only) PE-LNPs were also prepared to calculate the FRET efficiency E1 according to the following equation:
where ID and IDA are the donor fluorescence intensities at 530 nm of PE-LNPs labelled with the donor NBD-PE only and PE-LNPs co-labelled with both the donor NBD-PE and the acceptor NBD-PE, respectively.
In another experiment, PE-LNP 11′-65′ were labelled with NBD-PE (4 mM) and Rhod-PE (4 mM) separately. Then, PE-LNP 11′-65′ (NBD-PE only) were mixed with PE-LNP 11′-65′ (Rhod-PE only) in equal volumes under shaking at 100 rpm for 5 min, 1 h and 2 h, respectively. The control DOTAP liposomes were also labelled with NBD-PE (4 mM) and Rhod-PE (4 mM) separately, and the molar concentration of DOTAP in the control liposomes was equivalent to that of DOTAP in the corresponding PE-LNPs. The blank PE-LNPs and DOTAP liposomes without labelling were prepared for comparison. The FRET efficiency E2 at the specified time point was calculated according to the following equation:
where ID is the donor fluorescence intensity at 530 nm of PE-LNPs labelled with NBD-PE after mixing with blank samples, and IDM is the donor fluorescence intensity of PE-LNPs labelled with NBD-PE after mixing with PE-LNPs labelled with Rhod-PE.
Results and DiscussionTo further verify that lipid nanoparticles are formed in the core of nanostructures, FRET analysis using the NBD-PE and Rhod-PE pair was applied to investigate the spatial arrangement of PEG-PCL and DOTAP. The efficiency of energy transfer between the donor fluorophore NBD (4.0 mM) and the acceptor fluorophore Rhod (4.0 mM) is dependent on their distance on the membrane layer. The most common structure formed by mixing an amphiphilic polymer with a lipid is a hybrid vesicular architecture with mixed membrane composition27-29. As shown in
Further work was then carried out to validate the formation of lipid nanoparticles in the core of PE-LNPs. PE-LNP 11′-65′ were labeled with the donor NBD-PE (4.0 mM) or the receptor Rhod-PE (4.0 mM) separately. Then, PE-LNP 11′-65′ (NBD) were mixed with PE-LNP 11′-65′ (Rhod) in an equal volume under shaking at 100 rpm for up to 2 h. The control DOTAP-358 liposomes, where the number (i.e., 358) denotes to an equivalent molar concentration of DOTAP present in PE-LNP 11′-65′, were also labeled with the donor NBD-PE (4.0 mM) or receptor the receptor Rhod-PE (4.0 mM) separately for comparison. After DOTAP-358 liposomes (NBD) were mixed with DOTAP-358 liposomes (Rhod) for incubation under the same condition, an immediate increase in the FRET efficiency to 17.1% was observed, followed by a gradual increase to 22.0% after 1 h (
Transfection efficiency of saRNA-loaded PE-LNPs (saRNA in the nanoparticle core) For the firefly luciferase (fLuc) assay, HEK293 cells were seeded in a 96-well plate at a density of 5×104/well and cultured in the DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin for 48 h to reach 60˜80% confluence before transfection. Following the removal of spent medium, 100 μL of fresh serum-free or complete DMEM medium was added to each well, which was then added in replicates of 5 with 10 μL of fLuc saRNA-loaded PE-LNPs at different N/P molar ratios and polymer/saRNA (w/w) weight ratios (equivalent to 1 μg mL-1 saRNA). After 4 h of incubation, the transfection medium was replaced with the fresh complete DMEM medium. The fLuc activity, expressed as relative light units (RLU), in 50 μL of medium from the transfected cells following 24 h of treatment with 50 μL of fLuc substrate was assayed using a GloMax® Microplate Reader (Promega).
Transfection efficiency of saRNA-attached PE-LNPs (saRNA on the nanoparticle surface) fLuc saRNA-attached PE-LNPs were prepared by method 2 (M2) for comparison.
Briefly, 650 μg of PEG5k-PCL10kand 400 μg of DOTAP were co-dissolved in 0.5 mL of THF. The resulting organic solution was quickly added to 1 mL of Rnase-free water and stirred for 1-2 min at room temperature. THF was then removed by rotary evaporation to obtain the blank PE-LNPs. After that, 10 μg of fLuc saRNA was added in the blank PE-LNP solution. After vortexing for 30 s, fLuc saRNA-attached PE-LNPs were obtained. The transfection efficiency of fLuc saRNA-attached PE-LNPs in the absence or in the presence of FBS was determined using the abovementioned method in Example 4. The PEI/fLuc saRNA complexes and fLuc saRNA-loaded DOTAP lipid nanostructures were prepared as controls.
Transfection Efficiency of saRNA-Loaded PE-LNPs and saRNA-Attached PE-LNPs after Storage at 4° C.
100 μL of Rnase-free PBS (10×) buffer (or Rnase-free water) was added to 900 μL of fLuc saRNA-loaded PE-LNP or fLuc saRNA-attached PE-LNP solution, and the resulting samples were storage at 4° C. HEK293 cells were then transfected with these samples which were freshly prepared or stored at 4° C. for 5 days and the transfection efficiency was measured following the abovementioned method in Example 4.
Results and DiscussionThe effects of PEG-PCL molecular weights, N/P molar ratios and polymer/saRNA(w/w) weight ratios on the transfection efficiencies of various PE-LNPs were evaluated in HEK293 cells.
RNA molecules are very unstable and can be readily hydrolyzed/degraded. Two different RNA-loading methods were employed to compare the stability of RNA loaded in the core and attached on the surface of PE-LNPs. As shown in
The inventors further evaluated the stability of RNA loaded in the core and attached on the surface of PE-LNPs after storage in RNase-free water or PBS at 4° C. As shown in
These results suggest that the PE-LNP systems show the favorable serum stability and high encapsulation of biological payloads, in particular readily hydrolyzed/degraded RNA molecules, in the nanoparticle core can provide the optimal protection. This is because the hydrophilic PEG corona of PE-LNPs can reduce the protein absorption and the hydrophobic PCL layer can protect RNA from the harsh external environment.
Example 5—In Vitro Transfection in Interferon-Competent HeLa Cells by saRNA-Loaded PE-LNPsTransfection efficiency of saRNA-loaded PE-LNPs fLuc encoded saRNA was formulated in the interior of PE-LNP 11-65 by the same method described above in Example 2. HeLa cells were seeded and transfected following the abovementioned method in Example 4, where a titration was performed by changing the saRNA dose to 0.1, 0.5, 1, 3, 5 and 7 μg mL−1, respectively.
Results and DiscussionIn addition to inefficient protein expression, clinical applications of RNA systems are restricted by their high innate immunogenicity. It is crucial to achieve a good balance between protein expression and innate immune response. Toll-like receptors (TLRs) 3, 7, 8 and 9 are intracellular sensors of nucleic acids residing in endoplasmic reticulum, endosomes and lysosomes. Upon detection of foreign nucleic acids, the intracellular TLRs activate various signalling pathways and trigger the production of cytokines that could lead to limited protein expression or even adverse effect on the patient. Since HeLa cells express TLR3, transfection efficiency on HeLa cells were evaluated to examine the ability of PE-LNPs to prevent recognition by the intracellular TLRs.
These results also show that the PE-LNPs can effectively deliver nucleic acid payloads into various cell types, including human embryonic kidney cells (Example 4), cancer cells (Example 5) and T lymphocyte cells (Example 6).
Example 6—PE-LNP Mediated Intracellular Delivery of mRNA to Suspension Jurkat CellsTo further demonstrate the ability of PE-LNPs to deliver different payloads to different cell lines, the inventors used various PE-LNPs for intracellular delivery of green fluorescent protein (GFP)-encoding messenger RNA (mRNA) to suspension Jurkat cells. The GFP mRNA-loaded PE-LNPs were prepared by the same method described above in Example 2 for saRNA-loaded PE-LNPs. Briefly, Jurkat cells were gently pipetted to get resuspended and then counted by a Vi-CELL XR Cell Viability Analyzer (Beckman Coulter, USA). Cells were washed twice and then resuspended at the density of 2×106 cells mL1 in pre-warmed (37° C.) un-supplemented serum-free RPMI-1640 medium. 1 mL of cell suspension was seeded into each well of a 12-well plate. Then, GFP mRNA-loaded PE-LNPs were added dropwise at 2 μg mRNA per well, immediately followed by gentle pipetting to mix the culture thoroughly. After incubation at 37° C. for 4 h, the cells were washed twice and resuspended in 1 mL complete RPMI-1640 medium for further incubation at 37° C. overnight. Some of the sample wells were used for the viability test through cell counting by a Vi-CELL XR Cell Viability Analyzer. The rest sample wells were washed with PBS twice and cells were resuspended in 100 μL PBS containing the LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Thermo Fisher, USA) and incubated for 30 min. After washing with PBS, each sample was observed by fluorescence microscopy (EVOS Floid Imaging System, Thermo Fisher) and quantitatively analysed by flow cytometry (Canto, BD, USA) to examine the GFP expression in viable Jurkat cells.
Results and Discussion500 μg of FITC, 800 μg of PEG5k-PCL10k and 400 μg of DOTAP were co-dissolved in 0.5 mL of THF, 10 μg of saRNA was dissolved in 1 mL of Rnase-free water. The organic and aqueous solutions were quickly mixed and the mixture was kept stirring for 1-2 min at room temperature. THF was removed by rotary evaporation and free FITC was removed by centrifugation in ultrafiltration centrifugal tube (MWCO=3000) at 3000 rpm for 15 min to obtain the saRNA- and FITC- coloaded PE-LNPs.
The cellular uptake mechanism of the nanoscale system was investigated by laser scanning confocal microscopy. A 2 mL amount of HEK293 cells (2×105 cells per dish) was seeded into a 35 mm glass-bottom culture dish and cultured for 24 h. After pre-incubation at 4° C. for 1 h, saRNA- and FITC-coloaded PE-LNPs (final saRNA concentration at 2 μg per dish) were added and cells were further incubated at 4° C. for 4 h. HEK293 cells were treated with saRNA- and FITC-coloaded PE-LNPs at 37° C. for 4 h as control. After treatment at 4° C. or 37° C., cells were washed with PBS and fixed with a 4% paraformaldehyde solution for 10 min, and the nuclei and lysosomes were stained with Hoechst 33342 (5 μg mL1) and LysoTracker-Red (50 nM), respectively for 5 min. The cells were then imaged using a Leica SP8 Inverted confocal microscope and the fluorescence colocalization of FITC and LysoTracker-Red was analysed by Image J.
To further investigate the mechanism of cellular uptake and intracellular transport of saRNA- and FITC-coloaded PE-LNPs, HEK293 cells were seeded at a density of 5×105/well in a 6-well plate for 24 h. Firstly, cells were pre-incubated with the following inhibitors, respectively for 1 h: chlorpromazine hydrochloride (10 μg mL−1), methyl-β-cyclodextrin (MβCD, 5 mM), filipin (5 μg mL−1), amiloride (1 mM), genistein (40 μg mL-1) and nystatin (40 μg mL−1). Then, the saRNA- and FITC-coloaded PE-LNPs (containing 2 μg saRNA) were added to each well and co-incubated with the inhibitors for another 1 h. Finally, cells were washed with pre-cooled PBS solution for three times and was analyzed by flow cytometry (Fortessa I).
The uptake and intracellular trafficking of saRNA- and FITC-coloaded PE-LNPs were also studied by confocal microscopy. A 2 mL amount of HEK293 cells (2×105 cells per dish) was seeded into a 35 mm glass-bottom culture dish and cultured for 24 h. After pre-incubation with MβCD (5 mM) for 1 h, saRNA- and FITC-coloaded PE-LNPs (containing 2 μg saRNA) were added to the culture dish and co-incubated with MβCD for 4 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde solution for 10 min, and the nuclei and lysosomes were stained with Hoechst 33342 (5 μg mL−1) and LysoTracker-Red (50 nM), respectively for 5 min. Finally, the cells were imaged using a Leica SP8 Inverted confocal microscope.
Results and DiscussionIt is crucial to design a nano-carrier which can release endocytosed biological molecules into the cytoplasm by endosomal escape before they are trafficked to lysosomes for degradation30 (
The biocompatibility of saRNA-loaded PE-LNPs was investigated using a hemolysis method. Briefly, defibrinated sheep erythrocytes (RBCs) were centrifuged at 1500×g for 10 min at 4° C. and washed with PBS for three times. The cell pellets were resuspended into a 5% (v/v) erythrocyte suspension with PBS. A 100 μL aliquot of different PE-LNPs containing 1 μg saRNA were added into 0.9 mL of the RBC suspension in a centrifuge tube. Treatment of the RBC suspension with deionized water was used as the positive control. After incubation at 37° C. for 1 h, the RBC suspension was centrifuged and 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance (A) was measured at 540 nm using a spectrofluorometer (GloMax® Discover Microplate Reader, Promega, USA). The relative hemolysis was calculated according to the following equation:
The cytotoxicity of saRNA-loaded PE-LNPs against HEK293 cells was measured using alamarBlue assay. HEK293 cells were seeded in a 96-well plate at a density of 5×104 cells/well. After incubation for 24 h, the cells were treated with various PE-LNPs loaded with 1 μg mL-1 saRNA for 4 h. Then, 10 μL of alamarBlue HS reagent (5 mg mL-1) was added to each well. According to the manufacturer's instructions, after further incubation for 4 h, the absorbance of each well at 570 nm was measured using a Spectrofluorometer (GloMax@ Discover Microplate Reader, Promega, USA). The cytotoxic effect was determined from the absorbance readings.
Results and DiscussionThe hemolytic activity and non-specific cell cytotoxicity are typical issues associated with cationic carriers due to their strong interaction with negatively changed cell membranes. It is interesting to note that the hemolysis rates of various PE-LNPs were all below 10% after 1 h of treatment (
Female BALB/c mice (Charles River, UK), 6-8 weeks of age, were placed into groups of n=5 and housed in a fully acclimatized room with free access to food and water. All animals were handled in accordance with the UK Home Office Animals Scientific Procedures Act of 1986 in accordance with an internal ethics board and a UK government-approved project and personal license. Animals were conceded an adaption time of at least 7 days before the beginning of the experiments. Mice were injected intramuscularly (IM) in both hind leg quadriceps muscles with 5 μg of fLuc saRNA formulated in the interior of DOTAP lipid nanostructures (negative control), PE-LNP 5-65, PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively or formulated on the surface of PE-LNP 18-80 M2 (prepared with Method 2). After 7 days, the mice were injected intraperitoneally with 100 μL of XenoLight RediJect D-Luciferin Substrate (Perkin Elmer, UK) and allowed to rest for 10 min. Mice were then anesthetized using isoflurane and imaged on an In Vivo Imaging System FX Pro (Kodak Co., Rochester, NY, USA) equipped with Molecular Imaging Software Version 5.0 (Carestream Health, USA) for 10 min. A signal from each injection site was quantified using an equal detection area, using Molecular Imaging Software, and expressed as RLU.
In Vivo Immunogenicity of HA saRNA-Loaded PE-LNPs
Female BALB/c mice (Charles River, UK), 6-8 weeks of age, were placed into groups of n=5. Mice were immunized IM in one hind leg quadriceps muscle with 1 μg of saRNA encoding H1 hemagglutinin of the Cal/09 virus (HA saRNA) formulated in the interior of PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively to a total injection volume of 50 μL in 1×PBS and boosted with the identical formulation after 4 weeks. jetPEI/HA saRNA complexes were used as a control. Blood was collected after 4 and 6 weeks from study onset via tail bleeding. Blood was collected and centrifuged at 10,000×RPM for 5 min. The serum was harvested and stored at −80° C.
In vivo immunogenicity of SARS-CoV-2 saRNA-loaded PE-LNPs Female BALB/c mice (Charles River, UK), 6-8 weeks of age, were placed into groups of n=5. Mice were immunized IM in one hind leg quadriceps muscle with 1 μg of saRNA encoding a pre-fusion stabilized SARS-CoV-2 spike protein formulated in the interior of PE-LNP 11-65 and PE-LNP 18-65, respectively, to a total injection volume of 50 μL in 1×PBS. Blood was collected at week 4 from study onset via tail bleeding. Blood was collected and centrifuged at 10,000×RPM for 5 min. The serum was harvested and stored at −80° C.
HA-Specific ELISAHA antibody titers were quantitatively evaluated using the immunoglobulin ELISA protocol. Briefly, 0.5 μg mL−1 of HA-coated ELISA plate was blocked with 1% (w/v) bull serum albumin (BSA)/0.05% (v/v) Tween-20 in PBS. After washing, diluted serum samples were added to the plates, incubated for 2 h, and washed, and a 1:400o dilution of anti-mouse IgG-HRP (Southern Biotech, UK) was used. Standards were prepared by coating ELISA plate wells with antimouse Kappa (1:1000) and Lambda (1:1000) light chain (Serotec, UK), blocking with PBS/1% (w/v) BSA/0.05% (v/v) Tween-20, washing, and adding purified IgG (Southern Biotech, UK) starting at 1000 ng mL-1 and titrating down with a 5-fold dilution series. Samples and standards were developed using TMB (3,3′; 5,5′-tetramethylbenzidine), and the reaction was stopped after 5 min with Stop Solution (Insight Biotechnologies, UK). Absorbance was read on a spectrophotometer (VersaMax, Molecular Devices) with SoftMax Pro GxP v5 software.
SARS-CoV-2-Specific ELISASARS-CoV-2 antibody titers were quantitatively evaluated using the immunoglobulin ELISA protocol, following a similar procedure to the HA-specific ELISA.
Influenza ChallengeThree weeks after the boost injection, mice were challenged with 4.2×105 plaque forming units (pfu) of influenza (Cal/09) suspended in 100 μL of PBS. Mice were anesthetized using isoflurane, challenged intranasally (IN), and weighed each day to determine weight loss. According to the challenge protocol humane end-points, mice were euthanized if they sustained more than 3 days of 20% weight loss or 1 day of 25% weight loss.
Results and DiscussionTo study the in vivo saRNA expression efficiency, luciferase saRNA was formulated in various PE-LNPs and administered to mice by intramuscular injection with only one dose (5 μg/leg). After 7 days, the mice were imaged and the relative fluorescence intensity was quantified. The signal of saRNA/DOTAP lipid nanostructure-treated mice was hardly detectable, which might be due to the limited saRNA encapsulation ability and poor stability of saRNA/DOTAP lipid nanostructure. However, all the PE-LNP groups exhibited protein expression. Compared with PE-LNP 5-65, the three formulations PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 showed significantly higher luciferase expression (p<0.05) (
Furthermore, the inventors evaluated the immunogenicity and protective capacity of HA-encoding saRNA formulated in the interior of PE-LNPs after IM injection. One commercially available linear PEI, jetPEI which has previously been shown to effectively deliver RNA in vivo31, was used as a positive control. Mice received a prime and boost of 1 μg of saRNA formulated with jetPEI, PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80. The boost was administered 4 weeks after the initial prime. The mice were challenged IN with a Cal/09 influenza virus 2 weeks after the boost and weighed daily to monitor disease pathology (
The inventors also evaluated the immunogenicity of nCoV-encoding saRNA formulated in the interior of PE-LNPs after IM injection. Mice received a prime of 1 μg of saRNA formulated in PE-LNP 11-65 and PE-LNP 18-65, respectively. As shown in
PEG5k-PCL3-5k and PEG5k-PCL10k(Table 1) were synthesised and characterised following the same method described above in Example 1. saRNA was formulated in the interior of PE-LNP 11-65 with the amphiphilic polymers of different molecular weights using the same method described above in Example 2. The nano-formulations were stored in aqueous solution at 4° C. Their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL-1 at Day 0, 7 and 21 were then measured following the abovementioned method in Example 4.
Transfection Efficiency of Different saRNA-Loaded PE-LNPs after Storage in Aqueous Solution at 4° C.
saRNA was formulated in the interior of PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80 using the same method described above in Example 2. The nano-formulations were stored in aqueous solution at 4° C. Their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL-1 at Month 0 and 1 and 3 were then measured following the abovementioned method in Example 4.
Results and DiscussionAs shown in
As the PEG5k-PCL10k polymer was shown to be a superior option, a further investigation of the storage at 4° C. of liquid formulations of saRNA-loaded PE-LNPs without the presence of any stabilizing molecules was conducted.
Transfection efficiency of saRNA-loaded PE-LNP 11-65 after storage in aqueous solution at room temperature saRNA was formulated in the interior of PE-LNP 11-65 using the same method described above in Example 2. The nano-formulation was stored in aqueous solution at room temperature. Its in vitro HEK293 cell transfection efficiency at the saRNA dose of 1 μg mL-1 after storage at room temperature for 21 days was then measured and compared with the freshly prepared saRNA-loaded PE-LNP 11-65 following the abovementioned method in Example 4.
Transfection Efficiency of Different saRNA-Loaded PE-LNPs after Storage in Aqueous Solution at Room Temperature
saRNA was formulated in the interior of PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively, using the same method described above in Example 2. The nano-formulations were stored in aqueous solution at room temperature. Their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL−1 at Day 0, 14, 21 and 28 were then measured following the abovementioned method in Example 4.
Results and DiscussionAs shown in
Transfection Efficiency of Trehalose-Containing, saRNA-Loaded PE-LNP 11-65 after Storage in Aqueous Solution at 4° C.
The aqueous solution of saRNA (40 μg mL−1) and trehalose at the trehalose/saRNA (w/w) weight ratio of 100 was employed for the preparation of saRNA- and trehalose-coloaded PE-LNP 11-65 using the similar method as described above in Example 2. Then, additional trehalose was mixed with the obtained saRNA- and trehalose-coloaded PE-LNP 11-65 for topping up the total trehalose (both interior and exterior) to 250 mg mL-1.
As comparison, the aqueous solution of saRNA (40 μg mL−1) was formulated in the interior of PE-LNP 11-65 using the same method described above in Example 2. The obtained nano-formulation was then mixed with exterior trehalose at a concentration of 250 mg mL-1.
Those nano-formulations were stored in aqueous solution at 4° C. for 383 days, and then their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL−1 were measured and compared with the freshly prepared, saRNA-loaded, trehalose-free PE-LNP 11-65.
Results and DiscussionRNA molecules are very fragile and can readily degrade in exposed environments, thus requiring RNA vaccines and therapeutics to be stored and transported in a very challenging cold chain. Pfizer/BioNTech's mRNA vaccine, the world's first approved COVID-19 vaccine, is plagued by the major hurdle requiring storage at −70° C. Other RNA vaccines have the similar thermal stability issues, for example, Moderna's mRNA vaccine needs to be held at −20° C. for storage. This makes it extremely challenging for RNA vaccines to reach the required speed and scale of deployment to ensure herd immunity.
PE-LNPs formulated with RNA vaccines exhibited efficient in vivo protein expression and excellent immunogenicity in Example 9 described above (
To further prolong the shelf life of the RNA nano-formulations in aqueous solution, the exterior and/or interior stabilizing molecules such as trehalose was included (
Transfection Efficiency of saRNA- and Trehalose-Coloaded PE-LNPs after Storage in Aqueous Solution at Room Temperature
The aqueous solution of saRNA (40 μg mL−1) and trehalose at the trehalose/saRNA (w/w) weight ratio of 100 was employed for the preparation of saRNA- and trehalose-coloaded PE-LNP 11-65, PE-LNP 16-65 and PE-LNP 18-80, followed by mixing with additional trehalose for topping up to the total trehalose at 250 mg mL-1, using the same method as described above in Example 12.
Those nano-formulations were stored in aqueous solution at room temperature, and then their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL−1 at Day 0, 14, 21 and 28 were measured and compared with the freshly prepared, saRNA-loaded, trehalose-free PE-LNP 11-65, PE-LNP 16-65 and PE-LNP 18-80, respectively.
Results and DiscussionThe inventors then proceed to examine the effect of trehalose as a stabilizing molecule in the aqueous solution for storage at room temperature (
Optimization of Lyophilization Conditions for saRNA-Loaded PE-LNPs in the Presence of Exterior Trehalose
Different concentrations of saRNA (e.g., 20, 40, 60 and 100 μg mL-1) were formulated in the interior of PE-LNP 11-65 using the method described above in Example 2. The obtained nanoparticles were then mixed with exterior trehalose at a fixed concentration of 200 mg mL-1. The formulations were frozen in a −800C freezer, lyophilized for 48 h and then immediately rehydrated with RNase-free water. The DLS particle size distribution and in vitro HEK293 cell transfection efficiency of the rehydrated PE-LNPs at the saRNA dose of 1 μg mL−1 were evaluated to optimise the saRNA concentration during freeze-drying. After that, PE-LNP 11-65 loaded with the optimized saRNA concentration but mixed with different exterior trehalose concentrations (e.g., 150, 200, 250, 300 and 400 mg mL-1) were lyophilized and immediately rehydrated with RNase-free water for further analysis by DLS and in vitro transfection. Accordingly, the optimal concentrations of the loaded saRNA and the mixed trehalose for lyophilization of PE-LNP 11-65 formulations were identified.
Storage at 4° C. of Lyophilized saRNA-Loaded PE-LNPs saRNA in the Presence of Trehalose
The saRNA-loaded PE-LNP 11-65 containing 250 mg mL−1 exterior trehalose, as well as the saRNA- and trehalose-coloaded PE-LNP 11-65 (trehalose/saRNA weight ratio=100 for co-encapsulation, followed by mixing with additional trehalose for topping up the total trehalose to 250 mg mL-1) were lyophilized and then stored at 4° C. After that, the lyophilized PE-LNP formulations were rehydrated with RNase-free water for measurements of their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL-1.
Optimization of Lyophilization Conditions for saRNA- and Trehalose-Coloaded PE-LNPs
The aqueous solution of saRNA (fixed at 40 μg mL−1) and trehalose in RNase-free water at various trehalose/saRNA (w/w) weight ratios (e.g., 25, 50, 100, 200, 400 and 6250) was employed for the preparation of saRNA- and trehalose-coloaded PE-LNP 11-65 using the method described above in Example 2. Then, additional trehalose was mixed with the obtained saRNA- and trehalose-coloded PE-LNP 11-65 for topping up the total trehalose (both interior and exterior) to 250 mg mL-1 which was optimized from the abovementioned experiment in Example 14. In the particular case of saRNA- and trehalose-coloaded PE-LNP 11-65 with the trehalose/saRNA (w/w) ratio at 6250 and the saRNA concentration at 40 μg mL−1, there was no need to add additional trehalose for topping up. After lyophilization and immediate rehydration with RNase-free water, the colloidal stability of the saRNA- and trehalose-coloaded PE-LNP 11-65 was tested by DLS and their in vitro transfection efficiency at the saRNA dose of 1 μg mL−1 was analysed.
Results and DiscussionAnother strategy that the inventors have utilized for stable storage of RNA vaccines and therapeutics without the need for a very challenging cold chain is lyophilization of RNA-loaded PE-LNPs in the presence of exterior and/or interior stabilizing molecules such as trehalose (
First, PE-LNP 11-65 containing a fixed concentration of exterior trehalose at 200 mg mL−1 and various concentrations (20-100 μg mL−1) of saRNA loaded in the nanoparticle interior were lyophilized and immediately rehydrated with RNase-free water. The transfection efficiency of the rehydrated PE-LNP 11-65 were then evaluated. Interestingly,
Further, PE-LNP 11-65 containing the fixed interior saRNA concentration (40 μg mL-1) and various concentrations of exterior trehalose were lyophilized and immediately rehydrated with RNase-free water. According to
In order to further improve the RNA stability during storage, the inventors prepared the PE-LNP 11-65 coloaded with saRNA (at the fixed concentration of 40 μg mL-1) and trehalose (at different trehalose/saRNA weight ratios) in the hydrophilic core of the nanoparticles.
Additional trehalose was then mixed with the resulting saRNA- and trehalose-coloaded PE-LNP 11-65 for topping up the total trehalose (both interior and exterior) to 250 mg mL-1, followed by lyophilization of the formulations. It was found that when the trehalose/saRNA (w/w) weight ratio was above 400, the size of nanoparticles after lyophilization and rehydration with RNase-free water became bigger than 300 nm. The trehalose/saRNA weight ratio of 100 was the most favorable, leading to formation of the rehydrated PE-LNP 11-65 with the small uniform DLS size of 167.7 f 3.6 nm and the good monodispersity with a PDI of 0.313±0.017, comparable to the freshly prepared trehalose-free PE-LNP 11-65 (size=131.3±0.5 nm and PDI=0.265, shown in
Although the total trehalose concentration was fixed at 250 mg mL-1, it was favorable to increase the trehalose/saRNA (w/w) weight ratio up to 50-100 (i.e., increasing the proportion of the interior trehalose) for lyophilization of PE-LNP 11-65 formulations. As shown in
The optimized lyophilization conditions for saRNA- and trehalose-coloaded PE-LNP 11-65 (40 μg mL-1 saRNA and trehalose/saRNA weight ratio=100 for co-encapsulation, followed by mixing with additional trehalose for topping up the total trehalose to 250 mg mL-1) was chosen for the further heat burden study of the lyophilized formulations.
Example 15— Heat Burden Study of Lyophilized saRNA- and Trehalose-Coloaded PE-LNPs for Stable Storage at 40° C.The optimized saRNA- and trehalose-coloaded PE-LNP 11-65 formulations were lyophilized and then held for storage at 40° C. (tropical conditions). The saRNA-loaded PE-LNP 11-65 mixed with exterior trehalose was lyophilized as a control. After storage after a certain period, the lyophilized PE-LNP 11-65 formulations were rehydrated with RNase-free water and their in vitro transfection efficiencies were measured.
Results and DiscussionTo evaluate the potential of RNA- and trehalose-coloaded PE-LNPs for non-cold chain storage, the optimized saRNA- and trehalose-coloaded PE-LNP 11-65 were lyophilized and held for storage at 40° C. for 1, 3, 5, 7 or 14 days, respectively.
The inventors then utilized ultrafiltration centrifugation to remove free (unloaded) exterior trehalose after lyophilization, storage at 40° C. for 7 days and rehydration of saRNA- and trehalose-coloaded PE-LNP 11-65 and saRNA-loaded PE-LNP 11-65 mixed with exterior trehalose, respectively.
Using the method described above in Example 12, trehalose and 50 μg mL−1 calcein were pre-dissolved in the 40 μg mL−1 saRNA solution in RNase-free deionized water at the trehalose/saRNA (w/w) weight ratio of 100 for preparing saRNA-, trehalose- and calcein-coloaded PE-LNP 11-65, to which additional trehalose was mixed for topping up to the total trehalose (both interior and exterior) at 250 mg mL-1. saRNA- and calcein-coloaded PE-LNP 11-65 (trehalose-free) were prepared as a control. The uptake and intracellular trafficking of the two calcein-containing PE-LNP 11-65 formulations were investigated by laser scanning confocal microscopy. A 2 mL amount of HEK293 cells (2×105 cells per dish) was seeded into a 35 mm glass-bottom culture dish and cultured for 24 h. saRNA-, trehalose- and calcein-coloaded PE-LNP 11-65, or saRNA- and calcein-coloaded PE-LNP 11-65 were added to the culture dish (2 μg saRNA per dish). After treatment for 2 h, cells were washed and replenished with complete medium for a further 4 h of incubation. Cells were imaged using a Leica SP8 Inverted confocal microscope and mean fluorescence intensities of calcein in the confocal microscopy images were analyzed by ImageJ.
GFP-expressing mRNA- and trehalose-coloaded PE-LNP 11-65 (trehalose/mRNA weight ratio=100 for co-encapsulation, followed by mixing withb additional trehalose for topping up to the total trehalose at 10 mg mL1) were also prepared using the method described above in Example 12. Trehalose-free mRNA-loaded PE-LNP 11-65 were prepared as a control. HEK293 cells were seeded in a 6-well plate at 5×105/well and cultured for 48 h, and the in vitro transfection was quantitatively analysed by flow cytometry (Canto, BD, USA).
Results and DiscussionThe considerably enhanced transfection efficiencies of the saRNA-loaded PE-LNPs due to the presence of exterior/interior trehalose, as demonstrated in
The effect of trehalose on intracellular delivery of GFP-encoding mRNA to HEK293 cells by PE-LNP 11-65 was also investigated. As shown in
saRNA was formulated in the interior of PE-LNP 11-65 in presence of cholesterol following a similar method as described above in Example 2, where cholesterol was co-dissolved in THF with DOTAP and PEG-PCL. The cholesterol content (wt %) was defined as the weight percentage of cholesterol relative to cholesterol and DOTAP. The size, PDI and zeta potential of the nanoformulations were evaluated using a Litesizer (Anton Paar, UK). Their in vitro HEK293 cell transfection efficiencies at the saRNA dose of 1 μg mL1 were measured following the abovementioned method in Example 4.
Results and DiscussionThe preparation method for the PE-LNP system is readily adaptable to incorporate various lipids and their combinations to the nanoparticle core, including but not limited to cationic/ionizable lipids with chargeable groups and sterols such as cholesterol. The inventors chose cholesterol, to be formulated in the PE-LNP system, to further demonstrate the versatility of the nano system in its lipid compositions.
The inventors have developed polymer-enveloped lipid nanoparticles (PE-LNPs) to achieve efficient intracellular delivery of biological molecules including RNA both in vitro and in vivo and enable stable storage of vaccines and therapeutics without the need for a cold chain. This nano-formulation platform has the characteristics of an ideal carrier, including the favorable safety profile, compact size, controlled charge, high loading efficiency, efficient endosomolytic activity, superior colloidal and payload (RNA) stability, as well as simple, cost-effective and readily scalable preparation method.
PE-LNPs are composed of two main structural components: amphiphilic polymers such as PEG-PCL and cationic or ionizable lipids such as DOTAP, which have been proven to be biocompatible and approved by FDA. PE-LNPs can be prepared using a simple one-pot method through easy and rapid mixing and organic solvent evaporation. Cryo-TEM and FRET analysis have shown that the interior structure of PE-LNPs consists of lipid nanostructures, which are favorable for efficient payload encapsulation. The self-assembled PEG-PCL outer layer surrounding the interior lipid nanostructures can ensure colloidal stability and serum compatibility, and well protect the functionality of payloads such as extremely unstable RNA. PE-LNPs exhibited the excellent stability and high in vitro transfection efficiency, which can be four orders of magnitude higher than the commercially available PEI in the presence of FBS.
Mice transfected by IM injection with PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively showed strong luciferase expression. Furthermore, all mice were effectively protected in the Cal/09 influenza challenge after immunization with HA saRNA formulated in the interior of PE-LNP 11-65, PE-LNP 18-65 and PE-LNP 18-80, respectively because all of the nano-formulations induced high HA IgG antibody titers. The average weight loss of the mice immunized with HA saRNA-loaded PE-LNPs was less than 10%, especially PE-LNP 11-65 inducing the least amount of weight loss (˜8%).
The inventors have demonstrated two strategies for stable storage of biological payloads, in particular readily hydrolyzed/degraded RNA molecules, at ambient temperatures. The optimal protection offered by the core-shell nanoparticle structure enabled stable storage of RNA-loaded PE-LNPs in aqueous solution at room temperature. Another strategy for stable RNA storage is lyophilization of RNA-loaded PE-LNPs in the presence of exterior and/or interior stabilizing molecules such as trehalose. saRNA- and trehalose-coloaded PE-LNPs and saRNA-loaded PE-LNPs mixed with exterior trehalose showed the desired thermal stability of saRNA after lyophilization, storage at ambient temperatures as high as 40° C. (tropical conditions), and rehydration, with the former showing better performance. These demonstrate that the PE-LNP nano-formulations can enable stable storage of vaccines and therapeutics at ambient temperatures without the need for a cold chain. This offers a viable solution to improving global distribution of vaccine and therapeutic formulations.
Furthermore, the PE-LNPs developed by the inventors have been demonstrated to be generalizable to efficient intracellular delivery to different cell types and non-cold chain storage of various vaccines and therapeutics (based on biological molecules including but not limited to saRNA and mRNA).
MaterialsmPEG-OH (Mn=5,000 and 2,000), ε-caprolactone (ε-CL), toluene, diethyl ether, tetrahydrofuran (THF), 4% paraformaldehyde solution, fluorescein isothiocyanate (FITC), Hoechst, LysoTracker (red), Triton X-10o, bull serum albumin (BSA) and Tween-20 were purchased from Sigma-Aldrich. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, chloride salt) was bought from Avanti Polar Lipids. Trypsin-EDTA (0.25%, w/v), fetal bovine serum (FBS) and 1% penicillin/streptomycin were bought from Gibco (CA, USA). ONE-Glo™ Luciferase Assay System was obtained from InvivoGen. Phosphate buffered saline (PBS) and DMEM medium were obtained from Hyclone Laboratories (UT, USA). RNase-free water, RNase-free PBS (10×) and TPCK-trypsin were purchased from Thermo Fisher Scientific (UK). Trehalose Assay Kit was purchased from Abbexa. XenoLight RediJect D-Luciferin Substrate was bought from Perkin Elmer. Firefly luciferase saRNA and saRNA that encodes the H1 hemagglutinin of the Cal/09 virus were both kindly gifted by Prof. Robin Shattock's group at St Mary Hospital and Department of Infectious Disease, Imperial College London.
Cell CultureHEK293 (human embryonic kidney cell line) cells and HeLa (human cervical cancer cell line) cells were obtained from the ATCC (American Type Culture Collection, Wesel, Germany) and were cultured in high glucose DMEM medium (Gibco) supplemented with 10% (v/v) FBS (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Jurkat human T lymphocyte cells (Clone E6-1, ATCC® TIB-152™) were cultured in either RPMI-1640 or OPTIMEM medium supplemented with 10% (v/v) FBS, 100 U mL1 penicillin, 100 μg mL1 streptavidin and 2 mM L-glutamine (Rio medium). Cells were incubated in a humidified incubator with 5% CO2 at 37° C.
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Claims
1. A sub-micron particle comprising a payload molecule and a lipid structure being surrounded by an outer layer comprising an amphiphilic copolymer.
2. The sub-micron particle of claim 1, wherein the sub-micron particle has a largest maximum dimension of less than 1 μm.
3. The sub-micron particle of claim 1, or claim 2, wherein the payload molecule is a biomolecule and/or an active pharmaceutical ingredient (API).
4. The sub-micron particle of claim 3, wherein the biomolecule is a nucleic acid, and the nucleic acid is DNA, RNA or a DNA/RNA hybrid sequence.
5. The sub-micron particle of claim 4, wherein the RNA is self-amplifying RNA (saRNA) or messenger RNA (mRNA).
6. The sub-micron particle of any preceding claim, wherein the sub-micron particle comprises a plurality of lipid structures.
7. The sub-micron particle of any preceding claim, wherein the lipid structure is a lipid nanoparticle or a liposome, and preferably is a lipid nanoparticle.
8. The sub-micron particle of any preceding claim, wherein lipid structure comprises a cationic or ionizable lipid.
9. The sub-micron particle of any preceding claim, wherein the sub-micron particle has an N/P molar ratio of between 1:50 and 1,000:1, between 1:10 and 500:1, between 1:5 and 250:1, between 1:2 and 100:1, between 1:1 and 50:1, between 2:1 and 40:1, between 5:1 and 30:1, between 8:1 and 28:1, between 10:1 and 26:1, between 12:1 and 24:1, between 14:1 and 22:1 or between 16:1 and 20:1.
10. The sub-micron particle of any preceding claim, wherein the weight ratio of the amphiphilic copolymer to the payload molecule is between 5:1 and 1000:1, between 10:1 and 500:1, between 20:1 and 250:1, between 30:1 and 200:1, between 40:1 and 150:1, between 50:1 and 125:1, between 55:1 and 100:1, or between 60:1 and 85:1.
11. The sub-micron particle of any preceding claim, wherein the amphiphilic copolymer comprises at least one hydrophilic portion and at least one hydrophobic portion, and the hydrophilic portion comprises between 5 and 60 wt % of the amphiphilic copolymer and the hydrophobic portion comprises between 40 and 95% of the amphiphilic copolymer.
12. The sub-micron particle of any preceding claim, wherein the amphiphilic copolymer may have a molecular weight of between 1,000 and 100,000 Da.
13. The sub-micron particle of any preceding claim, wherein the weight ratio of the amphiphilic copolymer to the cationic or ionizable lipid is between 1:10 and 50:1, between 1:8 and 20:1, between 1:6 and 15:1, between 1:4 and 10:1, between 1:2 and 8:1, between 1:1.5 and 6:1, between 1:1 and 5.5:1, between 1.5:1 and 5:1, between 1.75:1 and 4.5:1, between 2:1 and 4:1, between 2.2:1 and 3.5:1, between 2.4:1 and 3:1, or between 2.5:1 and 2.7:1.
14. The sub-micron particle of any preceding claim, wherein the sub-micron particle further comprise at least one stabilizing molecule.
15. The sub-micron particle of claim 14, wherein at least one stabilizing molecule may be surrounded by the outer layer comprising the amphiphilic copolymer.
16. The sub-micron particle of claim 14, or claim 15, wherein the weight ratio of the at least one stabilizing molecule to the payload molecule is between 1:1 and 100,000:1, between 2:1 and 50,000:1, between 4:1 and 10,000:1, between 6:1 and 5,000:1, between 8:1 and 1,000:1, between 10:1 and 500:1, between 20:1 and 400:1, between 30:1 and 350:1 or between 40:1 and 300:1.
17. The sub-micron particle of any one of claims 14 to 16, wherein at least one stabilizing molecule may be disposed outside the outer layer comprising the amphiphilic copolymer, preferably wherein the at least one stabilizing molecule disposed outside the outer layer comprising the amphiphilic copolymer is disposed at a concentration of between 1 and 100,000 mg/ml, between 5 and 50,000 mg/ml, between 10 and 10,000 mg/ml, between 25 and 5,000 mg/ml, between 50 and 1,000 mg/ml, between 100 and 750 mg/ml, between 150 and 500 nm/ml, between 200 and 300 nm/ml, between 220 and 280 nm/ml, or between 240 and 260 mg/ml.
18. The sub-micron particle of any one of claims 14 to 17, wherein the or each stabilizing molecules is a carbohydrate and/or a polyol, preferably wherein the carbohydrate is a monosaccharide, a disaccharide, a trisaccharide, a polysaccharide, starch, cellulose or a polyol, and preferably is trehalose or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, stereoisomer or polymorphic form thereof.
19. A method of producing a sub-micron particle, the method comprising contacting a payload molecule, a cationic or ionizable lipid, and an amphiphilic copolymer to produce the sub-micron particle.
20. The method of claim 19, wherein the method comprises:
- providing a first solution comprising the cationic or ionizable lipid and the amphiphilic copolymer and an organic solvent;
- providing a second solution comprising the payload molecule and water;
- combining the first and second solutions to produce a reaction mixture, and thereby contacting the payload molecule, the cationic or ionizable lipid, and the amphiphilic copolymer.
21. The method of claim 19, or claim 20, wherein contacting the payload molecule, the cationic or ionizable lipid and the amphiphilic copolymer simultaneously, comprises contacting the payload molecule, the cationic or ionizable lipid, the amphiphilic copolymer and at least one stabilizing molecule.
22. The method of any one of claims 19 to 21, wherein the method comprises contacting the resultant sub-micron particle and at least one stabilizing molecule.
23. A sub-micron particle obtained or obtainable by the method any one of claim 19 to 22.
24. A pharmaceutical composition comprising the sub-micron particle of any one of claims 1 to 18 or 23 and a pharmaceutically acceptable vehicle.
25. A method of preparing the pharmaceutical composition according to claim 24, the method comprising contacting the sub-micron particle of any one of claims 1 to 18 or 23 with a pharmaceutically acceptable vehicle.
26. The sub-micron particle of any one of claims 1 to 18 or 23, or the pharmaceutical composition of claim 24, for use as a medicament.
27. A vaccine composition comprising the sub-micron particle of any one of claims 1 to 18 or 23, or the pharmaceutical composition of claim 24.
28. The sub-micron particle of any one of claims 1 to 18 or 23, the pharmaceutical composition of claim 24 or the vaccine of claim 27, for use in stimulating an immune response in a subject.
Type: Application
Filed: Jun 14, 2022
Publication Date: Sep 26, 2024
Inventors: Rongjun Chen (London), Xuhan Liu (London), Robin Shattock (London), Anna Blakney (London), Yifan Liu (London)
Application Number: 18/570,611
